Bag assembly for cultivation of cells

ABSTRACT

Provided is a bioreactor assembly that includes a plurality of cultivation bags at least one flexible temperature control bag having a thermostating fluid, and a tray adapted to rock back and forth around at least one axis. The flexible temperature control bag is mounted on the tray and the cultivation bags are located on the flexible temperature control bag.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation in Part of U.S. application Ser. No. 15/306,691, filed on Oct. 25, 2016, which is a filing under 35 U.S.C. 371 of international application number PCT/EP2015/059054, now International Publication No. WO2015/180908, filed on Apr. 27, 2015, which claims priority to U.S. application No. 62/003,754, filed May 28, 2014, the entire disclosures of each of which are hereby incorporated by reference in their entireties.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to bioreactors for cell cultivation, and more particularly to flexible bag bioreactors suitable for multi-stage expansion of cell cultures, such as seed train expansion or expansion of cells for cell therapy. The invention also relates to a method of expanding cell cultures in flexible bag bioreactors.

BACKGROUND OF THE INVENTION

When cell cultures are scaled up from a small cell bank sample to larger production batches, this normally has to be made in several steps, using separate bioreactors. This sequence of cultivations is often called a seed train and is needed in order to keep the cell densities within a certain optimal window, often in the order of>10⁵ cells per ml. In large scale production of biopharmaceuticals where the scale-up is from a small cryopreserved vial sample up to several m³, the seed train can involve up to six steps and take several weeks. It is also a complex procedure in that sterile transfer of the culture from one bioreactor to another is needed, such that transfers have to be made in LAF benches or sterile cleanrooms. Even under these conditions there is also a certain risk for adventitious infections, which can have disastrous consequences for a high value large scale culture. Similar concerns apply also for expansion of cells, e.g. stem cells, to be used in clinical cell therapy.

With the general trend towards single-use vessels for cell culture, there is an increasing tendency towards using flexible bag bioreactors in seed trains. However, the need to empty one smaller bag and to transfer the content into a larger bag still remains and is a work-intensive operation with some risk of contamination. It has been suggested to gradually increase the culture volume in flexible bags either by clamping off part of the bag over the entire bag cross section and then removing the clamp (WO2008153401) or by starting with a folded bag and then unfolding it ((US20100055764). However, these solutions do not provide good sealing between the used and unused compartments, leading to leakage of culture into the unused compartments and contamination of the cell culture by substances released from cells grown under unsuitable conditions. These methods also involve a substantial risk of mechanical damage to the bags, with resulting risks of bag rupture.

Accordingly, there is a need for a safe and convenient way of transferring cell cultures from one flexible culture compartment to another under sterile conditions. There is also a need for accurate temperature control of sensitive cell cultures, particularly in small volumes.

SUMMARY OF THE INVENTION

One aspect of the invention is to provide a flexible bag assembly which allows transfer of a cell culture from one cultivation compartment to another without risk of premature leakage between compartments or of bag damage. This is achieved with an assembly as defined in claim 1.

One advantage is that the cultivation compartments can be conveniently placed on a single tray and agitated using the same agitation means. A further advantage is that the culture can be easily transferred using e.g. gravity or gas pressure.

A second aspect of the invention is to provide a bioreactor assembly with flexible bags on a rocking platform, which allows transfer of a cell culture from one cultivation compartment to another without risk of premature leakage between compartments or of bag damage. This is achieved with an assembly as defined in the claims.

A third aspect of the invention is to provide a cultivation method where a cell culture is safely and conveniently transferred from one cultivation compartment to another. This is achieved with a method as defined in the claims.

A fourth aspect of the invention is to provide a flexible bag assembly which allows cultivation of cells in at least one cultivation compartment at substantially constant temperature. This is achieved with an assembly as defined in the claims.

A fifth aspect of the invention is to provide a bioreactor assembly which allows cultivation of cells in at least one cultivation compartment at substantially constant temperature. This is achieved with an assembly as defined in the claims.

A sixth aspect of the invention is to provide a cultivation method, where cells are cultivated at substantially constant temperature. This is achieved with a method as defined in the claims.

Further suitable embodiments of the invention are described in the dependent claims.

DRAWINGS

FIG. 1 shows an embodiment of the invention with three connectable bags arranged side by side on a tray.

FIG. 2 shows an embodiment of the invention with one bag having three connectable cultivation compartments.

FIG. 3 shows another embodiment of the invention with one bag having three connectable cultivation compartments.

FIG. 4 shows an embodiment of the invention with three connectable bags stacked on a tray.

FIG. 5 shows a sequence of cultivations in the three bags of FIG. 4, starting in a first bag at the bottom of the stack. a) cultivation in a first bag, b) cultivation in a second bag and c) cultivation in a third bag.

FIG. 6 shows an alternative sequence to FIG. 5, where the spent bag is removed before cultivation in the next bag. a) cultivation in a first bag, b) cultivation in a second bag and c) cultivation in a third bag.

FIG. 7 shows an alternative stacked bag arrangement with the first bag at the top of the stack. a) cultivation in a first bag, b) cultivation in a second bag and c) cultivation in a third bag.

FIG. 8 shows two examples of the bag-to-bag connection. a) dip tube drain port in a first bag connected with an inlet port in a second bag via tubing in a loop above the stack, b) drain port in a first bag connected with an inlet port in a second bag via tubing in a loop outside the stack.

FIG. 9. shows two examples of drain ports: a) a dip tube used with a bag in horizontal position and b) a port used with a bag in inclined position.

FIG. 10 shows an embodiment of the invention with one bag having three connectable cultivation compartments and a temperature control compartment.

FIG. 11 shows a variant of the FIG. 10 embodiment with a bag having one cultivation compartment and a temperature control compartment. The bag is shown in a) uninflated state, b) with the cultivation compartment inflated and c) with both compartments inflated.

FIG. 12 shows a bioreactor assembly of the invention with a separate temperature control bag and eight cultivation bags placed on top of the temperature control bag.

FIG. 13 shows a diamond-shaped cultivation bag.

FIG. 14 shows a gusseted cultivation bag. a) side view inflated, b) end view inflated, c) end view partially inflated, d) top view.

FIG. 15 shows a) cultivation bags with female and male connection tabs, b) cultivation bags connected by rods through the tabs, c) a bioreactor assembly with rod-connected cultivation bags on top of a temperature control bag and a tray, and d) an alternative bioreactor assembly with the rod-connected cultivation bags fixed to the temperature control bag by rod-loops.

FIG. 16 shows a cultivation bag strapped to the tray and the temperature control bag by adjustable straps.

FIG. 17 shows multiport fitments for cultivation bags, with a) two ports, b) three ports and c) four ports.

FIG. 18 a) shows an example of a branched connection to a port. FIG. 18 b) shows a tube inserted through a needleless valve into the cell culture in the bag.

FIG. 19 shows gas inlet and outlet port arrangements.

FIG. 20 shows arrangements for keeping bags in a raised/inflated state. a) Frame with (e.g. magnetic) retainers, b) Bag with tabs and frame with slit-type retainers, c) Bag with tabs/flaps and frame with ratchet bars.

FIG. 21 shows an additional arrangement for keeping bags in a raised/inflated state, with the bag top supported via a port and fitting.

DETAILED DESCRIPTION OF EMBODIMENTS

In one aspect, illustrated by FIGS. 1-10, the present invention discloses a flexible bag assembly 1;21;41;61;81 for cultivation of cells. The assembly comprises one or more bags 2,3,4;22;42;62,63,64,82, such as one or more flexible and/or collapsible bags, which form a plurality of cultivation compartments 5,6,7;25,26,27;45,46,47;65,66,67,85,86,87, such as two, three or more compartments. A first drain port 8;28;48;68;88 in at least a first cultivation compartment 5;25;45;65;85 is adapted to be fluidically connected with a second cultivation compartment 6;26;46;66;86 upon opening of a first valve means 10;30;50;70;90. The second cultivation compartment may be larger than the first cultivation compartment, e.g. having a volume of at least 120%, such as at least 150% or 120-1000% of the volume of the first cultivation compartment. The drain port can suitably be located at a low point of the first cultivation compartment to facilitate complete draining of the compartment. A low point can here mean a point which is located within the lowermost 20%, 10% or 5% of the compartment volume, either when the compartment is in a cultivation position or when it is moved to a transfer or drainage position. Two examples of drain ports are shown in FIG. 9, with FIG. 9 a) showing a dip tube 68 that can be used with a bag in cultivation position and FIG. 9 b) showing a plain port 68 at the end of a bag, which is suitable for draining when the bag is in an inclined or vertical drainage position with the port at the lower end of the bag. As shown in FIG. 8 a), it is also possible to use a dip tube at the end of the bag as a drain port 77. This arrangement allows draining in both horizontal and inclined/vertical positions. The valve means can be in a closed position when cells are cultured in the first cultivation compartment and then moved/rearranged to an open position when the culture is to be transferred to the second compartment. It can then optionally be moved/rearranged to a closed position to prevent leakage back to the first compartment during cultivation in the second cultivation compartment. Alternatively, the valve means may also comprise a check valve (not shown), only allowing flow in the direction from the first to the second compartment. The valve means can be a valve, such as e.g. a pinch valve, a diaphragm valve, a disc valve, a ball valve, a gate valve, a needle valve or a piston valve but it can also be a clamp, e.g. a pinch clamp, or it can be a breakable closure, e.g. a rupture disc or a breakable weld between the compartments. The valve means can be reversible, i.e. it can be moved/rearranged back to a closed position from an open position or it can be irreversible, i.e. it can only be moved/rearranged from a closed position to an open position. Valves and clamps are examples of reversible valve means and breakable closures of irreversible valve means. Pinch valves and pinch clamps have an advantage from a sterility/contamination point of view in that they can be applied externally on tubing or conduits and do not have any wetted parts. Pinch valves or pinch clamps adapted for application on tubing or conduits of up to 10 mm inner diameter or up to 15 mm outer diameter are particularly suitable in that they are simple low cost components that provide efficient closure and opening. The function of the valve means is to prevent any leakage of the culture into the second compartment during cultivation in the first compartment, when in the closed position, and to allow easy flow from the first to the second compartment when in the open position. If the valve means is reversible, or comprises a check valve, the valve may also have a function of preventing backflow of the culture into the first compartment during cultivation in the second compartment, if so desired.

The bag(s) 2,3,4;22;42;62,63,64,82 may be manufactured from one or more flexible plastic films or laminates, joined and sealed e.g. by welding. The films/laminates may comprise polyolefins, such as e.g. polyethylenes and/or ethylene vinyl acetate copolymers, but also barrier layers, e.g. ethylene vinyl alcohol polymers and/or tear resistant layers of e.g. polyamides. The thickness of the films or laminates may e.g. be 50-300 micrometers, such as 100-250 micrometers.

The bag assembly of the invention can conveniently be arranged on a rocking tray 13;33;73;93 platform to provide agitation during cultivation. The tray can rock back and forth around an axis 16;36;76,96, e.g. placed somewhat below the tray. Rocking tray platforms suitable for this purpose are described e.g. in U.S. Pat. No. 6,190,913, which is hereby incorporated by reference in its entirety, and are commercially available as WAVE Bioreactor™ from GE Healthcare Bio-Sciences.

In certain embodiments, the flexible bag assembly 1;21;41;61;81 comprises at least three cultivation compartments and has a second drain port 9;29;49;89 in the second cultivation compartment 6;26;46;66,86, which is adapted to be fluidically connected with a third cultivation compartment 7;27;47;67;87 upon opening of a second valve means 11;31;51,91. The second drain port and the second valve means can be constructed as described above for the first drain port and first valve means. The third cultivation compartment may be larger than the second cultivation compartment, e.g. having a volume of at least 120%, such as at least 150% or 120-1000% of the volume of the second cultivation compartment, and/or at least 140%, such as at least 200% or 140-10 000% of the volume of the first cultivation compartment. This allows a safe and convenient three-step scale-up from the first, to the second and then to the third cultivation compartment.

In some embodiments, illustrated by FIGS. 1 and 4, the flexible bag assembly 1;61 comprises at least two bags, which are suitably connected by tubing 12. The tubing can be connected to a drain port 8,9 in a first bag and to a culture inlet 17 in a second bag. The valve means 10, 11 may be a) valve components connected via the tubing, b) valves integrated with the tubing or c) externally applied devices such as pinch clamps or pinch valves. All these valve means are capable of preventing leakage in a closed position and allowing flow in an open position. The bags can be adapted to be placed beside each other on a tray 13 as in FIG. 1 or they can be adapted to be stacked on top of each other on a tray 73 as in FIGS. 4-7. It is also possible to have a combination of bags placed beside each other and stacked on top of each other, e.g. with two or three bags beside each other as in FIG. 1 and one larger bag placed below or on top of the two or three bags. The arrangement beside each other has the advantage that the assembly is easily prepared and arranged on the tray and the operation is simple The stacked arrangement has the advantage that the tray area is more efficiently used. When bags are stacked, it is advantageous if the connections are made by tubing and the tubing extends in a loop outside or above the stack. This improves accessibility and allows for easy operation of the valve means if it is placed in the loop. The bags can e.g. be stacked as shown in FIGS. 5 and 6, with the second bag on top of the first bag and optionally a third bag (and further bags) on top of the second bag. This arrangement has the advantage that any sensors at the bottom side of the bags can be in contact with sensor contacts on the tray surface. In particular, a culture sequence as illustrated in FIG. 6, where the first bag is removed from the stack after transfer of the culture to the second bag and the second bag is optionally removed after transfer of the culture to a third bag, allows sensors in all the bags to be in contact with the same sensor contacts on the tray surface. This sequence also has the advantage that during cultivation the entire bag area is in contact with the tray surface, which facilitates heat transfer from a heating element located on the tray surface and improves the temperature control. Alternatively, the bags can be stacked as shown in FIG. 7, with the second bag below the first bag and optionally a third bag (and further bags) below the second bag. This arrangement allows for easier constriction of the connections, as the drain port of a previous bag can easily be connected to an inlet port on the free top surface of a subsequent bag via a tubing loop above the stack. Another advantage is that it is easier to remove samples from each bag from sample ports on the free top surfaces of the bags. If needed, sheet-formed heating elements may be interspersed with the bags in the stack to improve the temperature control. For all the stack arrangements, if a spent bag is to be removed from the stack after transfer of the culture to a subsequent bag, this can easily be done under maintained sterile conditions e.g. by hot sealing disconnection if the connecting tubing is thermoplastic (made from e.g. soft PVC or thermoelastomers). Equipment for hot sealing of thermoplastic tubing is readily available, e.g. under the name of Hot Lips Tube Sealer (GE Healthcare Bio-Sciences AB).

In certain embodiments, illustrated by FIGS. 2-3 and 10, the flexible bag assembly 21;41 comprises at least one multi-compartment bag 22;42;82 with cultivation compartments 25,26,27;45,46,47;85,86,87 connected or connectable via internal conduits 32;92. The internal conduits can suitably have internal (circle-equivalent) diameters measuring less than 10%, such as less than 5%, of the cross section of the bag. As shown in FIGS. 2 and 10, the compartments may be delimited from each other by e.g. walls or welded seams extending at least over a major part of the bag cross section, such as the entire cross section. The internal conduits 32;92 can then e.g. be the hollow portions of ports 28,29;88,89 in walls or short pieces of tubing inserted as ports 28,29;88,89 in weld seams. The valve means 30,31;90,91 can either be integrated in the conduits or applied externally as clamps or pinch valves. The internal conduits 32;92 may alternatively be parts of the bag cross section to which the walls or weld seams do not extend, i.e. the walls/weld seams only extend over a part of the bag cross section, such as over 90-99.9% or 95-99.5% of the bag cross section, leaving the remaining part of the cross section open to form the internal conduits 32;92. The valve means can in this case suitably be externally applied clamps, which can provide leak-free seals as they only have to be applied over a short distance.

In certain embodiments, illustrated by FIG. 10, the flexible bag assembly 81 further comprises at least one bag 82 with at least one temperature control compartment 98. The bag 82 can suitably comprise a top film 99, a bottom film 100 and a delimiting film 97, wherein the delimiting film delimits the temperature control compartment 98 from the cultivation compartments 85,86,87. The temperature control compartment can suitably comprise at least an inlet (not shown) for a thermostating fluid. It may further comprise an outlet (not shown) for the thermostating fluid. The thermostating fluid can e.g. be water or cell culture medium. The thermostating fluid, such as water, can be circulated through the temperature control compartment via the inlet and outlet from/to a thermostating appliance, e.g. a thermostate bath. Alternatively, the temperature control compartment may be adapted to be located in direct contact with at least one temperature control surface or heating element on the tray 93. In this case, the thermostating fluid, such as water or cell culture medium, need not be circulated but can act as a temperature buffer. If the temperature control fluid is cell culture medium, the temperature control compartment may be fluidically connectable to the first, second or third cultivation compartment via a drain port and a valve means (not shown) and can then be used as a further cultivation compartment in the same way as discussed above. The advantage of having a temperature control compartment is that, particularly in small cultivation compartments, the temperature may be difficult to control accurately enough by the conventional means of having a temperature sensor in the cultivation compartment and controlling the temperature with a temperature control (heating) surface on the tray using a feedback loop. With a temperature control compartment a better temperature stability can be obtained due to the heat capacity of the thermostating fluid. The effect of the temperature control compartment is most evident for cultivation compartments of volumes 1 L or less such as 250 mL or less, 100 ml or less, 50-100 mL, 50-250 mL or 50-100 mL. This applies in particular to the first cultivation compartment. The volume of the temperature control compartment may e.g. be at least 1 L, such as at least 2 L or at least 5 L or at least 10 times, such as at least 20 times the volume of the first cultivation compartment.

In some embodiments, illustrated by FIG. 3, the flexible bag assembly 41 comprises at least one multi-compartment bag 42 with cultivation compartments 45,46,47 connectable via at least one breakable seal 52. The breakable seal may e.g. be a weak weld, as disclosed in EP 2,226,058A1 or U.S. Pat. No. 4,519,499, which are hereby incorporated by reference in their entireties. The breakable seal can e.g. constitute the entire delimitation between two adjacent compartments. In this case the weld is broken by bursting or other mechanical action from the outside of the bag, such that a first compartment opens up into a second compartment and the second cultivation step is conducted in the joint compartment. An advantage of this arrangement is that no extra components are needed. A further advantage is that, although separate gas inlets 54 and outlets 55 for each compartment are shown in FIG. 3, the cultivation can be performed with only one gas inlet and one gas outlet for the entire bag with all the compartments.

In certain embodiments of the flexible bag assembly, each cultivation compartment comprises a gas inlet 14;34;54;74;94 and a gas outlet 15;35;55;75;95. These inlets and outlets may be equipped with sterile filters (not shown) to prevent infection/contamination of the culture and are used to supply e.g. air/oxygen to the culture and to remove gaseous metabolites, such as carbon dioxide. They can also be used to supply a gas pressure to transfer the culture liquid from one compartment to another. The gas may e.g. be supplied via a gas inlet, while either closing the gas outlet or simply relying on the outlet sterile filter to provide a sufficiently high back pressure for transport of the liquid. The cultivation compartment(s) may also comprise one or more of sampling outlets, inlets for culture medium and sensors for e.g. temperature, cell density, pH and concentrations of e.g. oxygen or metabolites.

The bag assemblies disclosed above can suitably be supplied preassembled and presterilized, such as by radiation sterilization, e.g. by gamma or electron beam irradiation. Suitably, all liquid-contact materials are selected to be radiation-stable and to give low levels of leachables also after irradiation. All materials can e.g. be of USP VI quality.

In a second aspect, illustrated by FIGS. 1-4, the invention discloses a bioreactor assembly, comprising the flexible bag assembly 1;21;41;61;81 as disclosed above mounted on a tray 13;33;53;73;93 adapted to rock back and forth around at least one axis 16;36;56;76;96. The rocking mechanism and the support for the tray are not shown in the figures, but are described in detail in U.S. Pat. No. 6,190,913 and V Singh: Cytotechnology 30(1-3), 149-158 (1999). The bioreactor may further comprise a cell culture in at least one of the cultivation compartments 5,6,7;25,26,27;45,46,47;65,66,67;85,86,87. The tray may be equipped with a temperature control (heating) surface in direct contact with at least one bag. It may further be equipped with sensor connectors in electrical contact with at least one bag. At least one of the cultivation compartments may be connected to a gas supply via a gas inlet and a sterile filter.

In a third aspect, the present invention discloses a method of cultivating cells. The method comprises the steps of:

-   a) providing the bioreactor as disclosed above; -   b) introducing culture media and cells in the first cultivation     compartment 5;25;45;65;85; -   c) cultivating the cells in the first cultivation compartment to     provide a first cell culture; -   d) opening a first valve means 10;30;50;70;90 to fluidically connect     the first cultivation compartment with the second cultivation     compartment 6;26;46;66;86; -   e) transferring the first cell culture to the second cultivation     compartment; -   f) introducing culture media to the second cultivation compartment,     and; -   g) cultivating the cells in the second cultivation compartment to     provide a second cell culture.

The cultivations in the first and second cultivation compartments can be made using methods well known in the art and described e.g. in V Singh: Cytotechnology 30(1-3), 149-158 (1999) or Clincke et al., Biotechnol. Prog., 2013, Vol. 29, No. 3. Air or other gases may be supplied via gas inlets 14;34;74;94 and excess air/gas together with gaseous metabolites (e.g. carbon dioxide) may be vented via gas outlets 15;35;75;95. The cultivation in the first compartment may be continued until a predetermined viable cell density (VCD) is reached, e.g. 1.0×10⁵, 2.0×10⁵, 5.0×10⁵ or 1.0×10⁶ viable cells/ml. The VCD may e.g. be measured with an inline biomass sensor, e.g. as described in U.S. Pat. No. 8,180,575 or WO 2010/010313A2, which are hereby incorporated by reference in their entireties. When the predetermined VCD has been reached, steps d) and further may be initiated. The transfer in step e) may be accomplished by gravity, e.g. by inclining the tray with the bags or raising it vertically. It may also be accomplished by gas pressure, suitable delivered via the gas inlet of the first bag, in which case the back pressure of a sterile filter on the gas outlet may be sufficient to allow the transfer. The transfer may further be accomplished by other means, e.g. application of pressure on the first or by combinations of different means.

In some embodiments, the method further comprises a step e′), after step e), of closing the first valve means. Alternatively, or additionally, the first and second compartments may be disconnected from each other e.g. by cutting and sealing any tubing 12;72 between the compartments. In the case where the compartments are located in separate bags, this enables removal of the first bag from the tray, which can improve heat transfer and/or allow contact with sensor contacts on the tray surface as discussed above.

In certain embodiments, the method further comprises the steps of:

-   h) opening a second valve means to fluidically connect the second     cultivation compartment with the third cultivation compartment; -   i) transferring the second cell culture to the third cultivation     compartment; -   j) introducing media to the third cultivation compartment, and; -   k) cultivating the cells in the third cultivation compartment to     provide a third cell culture.

This method can further comprise a step i′), after step i), of closing the second valve means. Alternatively, or additionally, the second and third compartments may be disconnected from each other e.g. by cutting and sealing any tubing 12;72 between the compartments.

In some embodiments, step k) and/or g) may be conducted in perfusion mode, i.e. by conveying at least part of the culture to a filter where a filtrate is removed and conveying the cells back to the culture and replacing the removed filtrate with fresh culture medium. This allows for further increases in the VCD.

In a fourth aspect, the present invention discloses a flexible bag assembly for cultivation of cells. The assembly comprises at least one bag 82 with at least one cultivation compartment 85,86,87 and at least one temperature control compartment 98. The temperature control compartment can suitably be adapted to rest on a tray support, with the cultivation compartment(s) above the temperature control compartment. The cultivation compartment(s) may be fluidically connectable as disclosed above, but they may also be separate and adapted for parallel cultivation. The bag 82 can, as illustrated by FIGS. 10 and 11, comprise a top film 99, a bottom film 100 and a delimiting film 97, wherein the delimiting film delimits the at least one temperature control compartment 98 from at least one cultivation compartment 85,86,87, such as each cultivation compartment. The cultivation compartment(s), such as each cultivation compartment, may e.g. have a volume of less than 1 L, such as 50-250 mL, and/or the volume of said temperature control compartment may e.g. be at least 10 times the volume of at least one cultivation compartment, such as of each cultivation compartment. The temperature control compartment may e.g. comprise an inlet and an outlet for a thermostating fluid to allow for circulation of thermostating fluid from e.g. a thermostat bath. Alternatively, the temperature control compartment may have an inlet for thermostating fluid to allow for cultivation under conditions where the temperature control compartment is closed and acts as a temperature buffer. The bag assembly can suitably be constructed from films or laminates such that the inner surfaces of the cultivation compartment(s) are amenable to cell cultivation without negative effects on cells. Typical film materials with cell-friendly surfaces include polyethylene (in particular ultra low density polyethylene) and ethylene-vinyl acetate copolymers. Such materials are discussed e.g. in US20150360450, US20150336363, U.S. Pat. No. 5,988,422 and U.S. Pat. No. 8,556,497, which are hereby incorporated by reference in their entireties. The use these film/laminate materials is also considered for the inner surfaces of all cultivation compartments and cultivation bags discussed above and below.

In a fifth aspect, the present invention discloses a bioreactor assembly comprising:

-   a) at least one bag 82 or bottle with at least one cultivation     compartment 85,86,87; -   b) at least one flexible temperature control compartment 98     comprising a thermostating fluid, and; -   c) a tray 93 or a stack of trays adapted to rock back and forth     around at least one axis 96.     The at least one flexible temperature control compartment is mounted     or placed on the tray and the at least one bag or bottle is suitably     located on top of the at least one flexible temperature control     compartment. Thus, the heat transfer between the thermostating fluid     and a cell culture in the cultivation compartment can proceed     efficiently and the heat capacity of the thermostating fluid     provides good temperature control within a narrow temperature range     even for small cultivation compartments, such as cultivation     compartments of volumes 1 L or less such as 250 mL or less, 100 ml     or less, 50-100 mL, 50-250 mL or 50-100 mL. The bag 82 may comprise     e.g. one, two, three, four, five, six, seven, eight, nine or ten     cultivation compartments, which can be adapted for parallel     cultivation and/or for sequential cultivation as disclosed above.     The flexible temperature control compartment may form a part of the     at least one bag 82, i.e. bag 82 may comprise both the flexible     temperature control compartment 98 and at least one cultivation     compartment 85,86,87, e.g. as illustrated in FIG. 10. The at least     one bag may further comprise a top film 99, a bottom film 100 and a     delimiting film 97, wherein the delimiting film delimits the at     least one temperature control compartment 98 from at least one     cultivation compartments 85,86,87. With this arrangement, an     integral bag with one or more cultivation compartments in a top part     of the bag and a temperature control compartment in a bottom part is     obtained. The temperature control compartment can be equipped with     an inlet and an outlet for thermostating fluid to allow circulation     of the fluid from e.g. a thermostat bath through the temperature     control compartment. One or more of the cultivation compartments,     such as each cultivation compartment, may also be equipped with     temperature sensors, which can be electrically or     electromagnetically connected to a control unit to allow temperature     control with a feedback loop. The control unit can further be     connected to a thermostat bath or other temperature control     equipment in a feedback loop. Further, or alternatively, the     temperature control compartment may comprise a temperature sensor,     which can be connected to the control unit. The tray may be equipped     with a temperature control (heating) surface in direct contact with     at least one bag. It may further be equipped with sensor connectors     in electrical contact with at least one bag. At least one of the     cultivation compartments may be connected to a gas supply via a gas     inlet and a sterile filter.

As illustrated in FIG. 12, the flexible temperature control compartment 98 a may be in the form of a separate temperature control bag 98 a (which can alternatively also be used as a secondary cultivation bag for scaling-up of the culture as discussed above) 98 a located on a tray 93 adapted to rock back and forth around at least one axis 96. One or more cultivation bags 82 a may then be placed on top of bag 98 a, for sequential and/or parallel cultivation as disclosed above. This arrangement allows a high degree of flexibility with regard to the size, number and type of the cultivation bags. The cultivation bags 82 a can be single-compartment bags or multicompartment bags as disclosed above, suitably with one or more inlet and outlet ports 94,95 per cultivation bag or cultivation compartment. Temperature control bag 98 a suitably contains a thermostating fluid (e.g. an aqueous liquid), the temperature of which may be controlled a) by circulation from e.g. a thermostat bath through the temperature control bag via an inlet 101 and an outlet 102 or b) by a heat control surface 103 between the temperature control bag 98 a and the tray 93 or integrated in a surface of the tray. The heat control surface may e.g. be an electrically heated surface. It is also possible to have a combination of fluid circulation and a heat control surface.

The cultivation bags may in many cases be relatively small, e.g. adapted to work with a culture (working) volume in the range of 30-500 ml, such as 30-100 ml. The culture volume is normally in the range of 10-20% of the total inflated bag volume, due to a) the need for an oxygen reservoir in the headspace and b) accommodation of proper wave-formed agitation in rocking bags. This means that the bag volumes may e.g. be in the range of 150-5000 ml, such as 150-1000 ml, 150-500 ml or 150-300 ml. In this case, small standard rectangular pillow-style two-dimensional bags (made from two sheets welded to each other along the four perimeter sides) have a low height, allowing only a small headspace height in the inflated bag. During agitation by rocking, this leads to a risk of liquid splashing up into the ports on the top of the bag and blocking e.g. the exhaust gas filter. This can be avoided if the headspace height is at least 5 cm, which translates to an inflated bag height h of at least 6 cm. One way to achieve this is with an elongated hexagonal bag 120, as depicted in FIG. 13. Here, the additional width W-w (the maximum width minus the minimum width) at the center of the bag allows an increased height h, while keeping the bag volume low. The bag typically comprises a top sheet 121 and a bottom sheet 122, joined to each other, e.g. by welding, along six substantially linear seams 123,124, together forming the perimeter of an elongated hexagon. The top and bottom sheets 121,122 together with the six seams 123,124 can define an inner volume 129 of the bag. Two seams are denoted end seams 123 and may be located opposite each other and be substantially parallel. Four oblique side seams 124 may each intersect one end seam and another side seam. The length-to-width ratio, L/W of the hexagon can suitably be at least 1.1, such as at least 1.5 or 1.5-3. It is also possible to add further side seams angled to each other, e.g. creating an elongated octagon with L/W at least 1.1, such as at least 1.5 or 1.5-3. The bag may further comprise channel-formed tabs 125, adjacent to the end seams 123, adapted to receive fixation rods 126 for fixation of the bag to a tray and/or to other bags. Also, the bag may comprise one or more, such as at least two, ports 127 located in the top sheet, suitably in a central region of the bag. The central region may e.g. be defined as a region where the shortest distance to an end seam is less than or equal to L/4. The ports can be used for addition and removal of gases, liquids and other materials before, during and after cultivation of cells in the inner volume 129 of the bag. Each port 127 can be comprised in one fitting 128 for attachment to the top sheet as shown in FIG. 13, but it is also possible to have more than one port, e.g. two ports, per fitting as discussed below.

Another way of achieving a low volume bag with high inflated height h is to use a three-dimensional gusseted bag 140, as illustrated in FIG. 14. Such a bag can be constructed from a top sheet 141, a bottom sheet 142 and two gusset sheets 143. They can be joined, e.g. by welding, along four side seams 144 and two end seams 145 to form a bag with an inner volume 146 defined by sheets 141,142,143 and seams 144,145. The gusset sheets may be folded over during welding, to form an essentially flat structure before inflation, where the gusset sheets unfold during inflation to form a bag of roughly rectangular octahedral shape of length L and height h. If desired, a gusseted bag of similar shape can also be constructed from a single sheet or a tubular structure, where three or four of the side seams are replaced by longitudinal folds of the sheet/tube. As discussed above, the bag volumes may e.g. be in the range of 150-5000 ml (or larger), such as 150-2000 ml or 150-1000 ml, meaning that they are adapted to work with culture (working) volumes e.g. in the range of 30-500 ml, such as 30-200 or 30-100 ml. The three-dimensional shape allows high headspace heights and also allows for accommodation of many bags on a given tray area through reduction of the bag footprint. Typical values of length L, width W and height h may e.g. be L 6-50 cm, such as 6-25 cm, W 5-30 cm, such as 5-15 cm, and h 5-20 cm, such as 5-10 cm. The bag may further comprise one or more, such as at least two, ports 147 suitably located in the top sheet, e.g. in a central region of the bag. The central region may e.g. be defined as a region where the shortest distance to an end seam is less than or equal to L/4. The ports can be used for addition and removal of gases, liquids and other materials before, during and after cultivation of cells in the inner volume 146 of the bag. Each port 147 can be comprised in one fitting 148 for attachment to the top sheet as shown in FIG. 14, but it is also possible to have more than one port, e.g. two ports, per fitting as discussed below.

When multiple cultivation bags 160,161 are to be secured in a rocking tray 162 on top of a temperature control bag (or a secondary cultivation bag) 98 a, a swift and easy fixation of the bags to each other and to the tray is needed. One way of achieving this is to use hollow tabs 163,164 and rods 165,166 as illustrated in FIG. 15. A combination of “female” 160 and “male” 161 cultivation bags can be used, where the male bags comprise hollow tabs 163 at the center of the end seams 167 and the female bags comprise two hollow tabs 164 at each end seam 168, located away from the center. When a male and a female bag are placed adjacent to each other, end seam to end seam, the tabs interleave, allowing a bag connector rod 165 inserted through the hollow tabs 163,164 to fix the bags to each other. Tray connector rods 166 inserted through the opposite tabs 163,164 of the bags and through loops or hooks 169 attached to the tray walls 170 can then be used to fix the bag package to the tray. For improved fixation to the temperature control bag, the bag connector rod 165 can also be inserted through film loops 171 attached (e.g. welded) to the top film 172 of the temperature control bag, as depicted in FIG. 15 d). This arrangement helps holding down the cultivation bags to the temperature control bag, which increases the contact area and improves the heat transfer.

Multiple cultivation bags 180 may also be secured in a tray 181 on top of a temperature control bag (or a secondary cultivation bag) 98 a by application of tensioners 182. The tensioners 182 can suitably be adjustable, such that bags 180 of several different sizes can be accommodated, with sufficient tension such that the bottom film 186 of the cultivation bag 180 is in intimate contact with the top film 187 of the temperature control bag 98 a, in order to provide efficient heat transport between the bags. The tensioners 182 may e.g. be Velcro (separable fabric hook and loop fasteners) straps, but many other designs are also possible, e.g. straps with buckles, strap adjusters etc, springs or elastic straps with hooks or screw adjustment tensioners. The tensioners may suitable be attached to the bag via bag fasteners 183, e.g. rigid loops attached to the bag end. The tensioners may further be attached to the tray via tray fasteners 185, which can e.g. be rigid loops that may be attached to the tray wall 184. Alternatively, the tray fasteners may be located on the outside of the tray or on the tray bottom.

For cell cultivation in bags it is desirable to have several ports in the bag, to allow filling with culture media, inoculation, gas entry and exit, sampling, sensing, harvesting etc. For small bags, as discussed above, the available area on the bag top can be a limiting factor, particularly for conventional fittings, with a circular plate for welding to the bag film and a single port in the center of the plate. The number of ports can however be increased by the use of fittings 202,203,204 with several ports in each fitting, as illustrated by FIG. 17. Here, two, three or four ports 200 are accommodated in plates 201 that can be welded to the bag film. One or more, e.g. two, such fittings may be mounted to the top 121;141 of a cultivation bag 120;140 to allow access through multiple ports.

It is also possible to achieve multiple entry points to a bag from a single port, as illustrated in FIG. 18. Here, a branched port 210 or a branched line to a port is used to allow e.g. simultaneous fluid entry/exit and/or sensing through a port 210 attached to the top film 211 of a cultivation bag. The branches 212,213 can e.g. be equipped with hose barbs 214 for attachment to (sterile) fluid lines and/or sanitary valves/connectors 215 for sampling, sensing or addition of fluids. The sanitary valve(s)/connector(s) may e.g. be swabable needleless valves as exemplified by the Clave ports available from NCU Medical Inc, swabable septum ports or aseptic connectors such as ReadyMate (GE Healthcare). The branching may be via a T-branch as shown in FIG. 18. With a sanitary valve/connector 215 on the vertical branch 213, it is then possible to access the cell culture medium 217 in the interior 216 of the bag via a needle or tubing 218 inserted through the valve/connector. Suitably, the sanitary valve/connector 215 and the vertical branch 213 are aligned such that a straight line passage is formed from the valve/connector to the interior of the bag 216 for insertion of needle/tubing 218 or a sensor. This is convenient for sampling and sensing applications. It is however also possible to have Y-branches and/or multiple branches, where one or more branches may be closed, e.g. by clamping until they are used.

Another effect of using small bags is that somewhat higher gas pressures than normal may be needed to keep the bag inflated. Bags are normally manufactured in a deflated (flat) configuration and the elasticity of the wall construction will resist the inflation. As the side wall-to-volume ratio decreases with decreasing volume, the inflation resistance will thus increase. One suitable way to increase the gas pressure is to add a constriction on a gas exhaust line. This can be done e.g. as shown in FIG. 19, where in a cultivation bag 220 with a gas inlet port 221 and a gas outlet port 222, a constriction 223 is added on the gas exhaust line 224 in addition to the sterile filters 225 normally used with cultivation bags. The constriction 223 may e.g. be an extra filter, but it can also be other types of constrictions known in the art, e.g. an adjustable valve.

An alternative way to handle the increased inflation resistance of small bags is to use a locking device or catch that, once the bag is inflated, locks it in an expanded configuration. As illustrated in FIG. 20, the bag 140 can have one or more fastener elements 240;241;242;257, e.g. attached to a top film 141, or to a top portion 260 of a side film/gusset sheet 143, of the bag. When the bag is inflated, the fastener element(s) can then engage with one or more retainer elements (which may also be called receiver elements) 245;246;247;256 located on a support device at a position corresponding to the inflated height h of the bag. The support device can e.g. be a rigid frame 250 with one or more base portions 251 and one or more support portions (e.g. pillars or walls) 252 extending in a vertical direction. The fastener elements 240;241;257 can be manually engaged with the retainer elements 245;246;256, e.g. by hook-and-loop action, tab-slit action, insert-receiver action, snap action, pressure sensitive adhesives etc. The fastener elements may be directly attached to the top film 141 or indirectly, e.g. via a port 147 and fitting 148 as illustrated in FIG. 21. Here, the fastener element 257 is a wide object, e.g. a filter, attached to the port and inserted in a receiver structure 256 on a top horizontal portion of frame 250. The fastener element 257 could in this case also be a barb or bulbous object attached to the port. Alternatively, the support device and the fastener and retainer elements may be arranged to provide automatic engagement once the bag is inflated to a predetermined level. This can e.g. be achieved by magnetic fastener elements 240 and retainer elements 245 (one of the fastener element or retainer element pair can be a permanent magnet and the other element of the pair can be either another permanent magnet or a ferromagnetic element). Automatic engagement can also be achieved in many other ways, e.g. by ratchet-type devices as illustrated in FIG. 20 c), where tabs or flaps 242 on the bag top engage with ratchet bars 247 on the support device.

In a sixth aspect, the present invention discloses a method of cultivating cells. The method comprises the steps of:

-   a) providing the bioreactor assembly as disclosed above; -   b) introducing culture media and cells in at least one cultivation     compartment 85,86,87 and; -   d) cultivating the cells in the at least one cultivation     compartment.

In certain embodiments, the temperature in step c) is maintained within a range of a target temperature +/−2° C., such as +/−1° C. or +/−0.5° C. The target temperature can depend on the cell type, and can for mammalian cells be e.g. 35-38° C., such as 36-37° C.

In some embodiments, the thermostating fluid is circulated through said flexible temperature control compartment. This allows for precise control of the temperature. Alternatively, the temperature control compartment is in contact with a temperature control (heating) surface on the tray and the temperature is controlled using a temperature sensor in/on the temperature control compartment using a control unit and a feedback loop.

In certain embodiments said at least one cultivation compartment comprises a temperature sensor which is electrically or electromagnetically connected to a control unit and the temperature of said thermostating fluid and/or said cultivation compartment is controlled by the control unit using a feedback loop.

This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims. All patents and patent applications mentioned in the text are hereby incorporated by reference in their entireties as if individually incorporated. 

The invention claimed is:
 1. A bioreactor assembly comprising: a) a plurality of independent cultivation bags, wherein the plurality of independent cultivation bags are fluidically connected in series through a respective valve between each adjacent pair of independent cultivation bags of the plurality of independent bags; b) one flexible temperature control bag having a top surface directly contacting a bottom surface of each independent cultivation bag of the plurality of independent cultivation bags, and configured to contain a thermostating fluid to facilitate heat transfer between the one flexible temperature control bag and a cell culture in the plurality of independent cultivation bags, and; c) a tray adapted to rock back and forth around at least one axis, wherein said flexible temperature control bag is mounted on said tray and said cultivation bags are seated on said top surface of the flexible temperature control bag, and wherein the cultivation bags are secured relative to the flexible temperature control bag through attachment to at least one of the flexible temperature control bag or the tray.
 2. The bioreactor assembly of claim 1, wherein said temperature control bag comprises an inlet and an outlet for the thermostating fluid.
 3. The bioreactor assembly of claim 1, wherein each cultivation bag comprises a gas inlet and a gas outlet.
 4. The bioreactor assembly of claim 1, wherein each cultivation bag comprises at least two hollow tabs adapted for insertion of a bag connector rod and a tray connector rod through said tabs.
 5. The bioreactor assembly of claim 1, wherein said flexible temperature control bag comprises a plurality of loops adapted for insertion of a bag connector rod for securing the cultivation bags.
 6. The bioreactor assembly of claim 1, wherein said cultivation bags are secured to said tray by tensioners.
 7. The bioreactor assembly of claim 6, wherein said tensioners comprise straps.
 8. The bioreactor assembly of claim 6, wherein said tensioners comprise separable fabric hook and loop fastener straps.
 9. The bioreactor assembly of claim 1, wherein the independent cultivation bag has an inflated volume of 150-1000 ml.
 10. The bioreactor assembly of claim 9, wherein the independent cultivation bag has an elongated hexagonal or octagonal shape.
 11. The bioreactor assembly of claim 9, wherein the independent cultivation bag is a three-dimensional gusseted bag.
 12. The bioreactor assembly of claim 11, wherein said three-dimensional gusseted bag has an inflated height of 5-10 cm.
 13. A flexible cell cultivation bag having an inflated volume of 150-1000 ml and an inflated height of 5-10 cm seated on top of a secondary cultivating bag being used as a flexible temperature control bag having a top surface in direct contact with a bottom surface of the flexible cell cultivation bag, configured to contain a thermostating fluid to facilitate heat transfer between the thermostating fluid and a cell culture in the flexible cultivation bag, wherein the cultivation bag is secured relative to the at least one flexible temperature control bag through attachment to at least one of the temperature control bag and a tray; and wherein the cultivation bag is fluidically connected to at least a third cultivation bag through a valve.
 14. The flexible cell cultivation bag of claim 13, equipped with at least two ports located in a top film of the cell cultivation bag.
 15. The flexible cell cultivation bag of claim 13, which is a three-dimensional gusseted bag.
 16. The flexible cell cultivation bag of claim 14, comprising at least one fitting with a plurality of ports in said fitting.
 17. The flexible cell cultivation bag of claim 14, comprising at least one branched port or branched line attached to a port.
 18. The flexible cell cultivation bag of claim 17, wherein at least one port branch is connected to a swabable needleless valve.
 19. The flexible cell cultivation bag of claim 13, comprising one or more fastener elements attached to a top film, or to a top portion of a side film, of said bag, wherein said fastener element or elements is/are arranged to engage with one or more retainer elements on a support device when the bag is inflated.
 20. The flexible cell cultivation bag of claim 19, wherein said fastener element or elements and retainer element or elements are arranged to automatically engage with each other upon inflation of the bag to a predetermined level.
 21. An assembly of a flexible bag according to claim 19, and a support device with one or more retainer elements arranged to engage with said fastener element or elements when the bag is inflated.
 22. An assembly of a flexible bag according to claim 13, comprising the secondary cultivation bag used as the flexible temperature control bag disposed on a tray adapted to rock back and forth around at least one axis.
 23. The bioreactor assembly of claim 1, further comprising a heat control surface disposed between the tray and the flexible temperature control bag, for facilitating heat temperature control.
 24. The bioreactor assembly of claim 1, wherein the plurality of independent cultivation bags comprises a first cultivation bag and a second cultivation bag, wherein the volume of the second cultivation bag is at least 120% of the volume of the first cultivation bag.
 25. The bioreactor assembly of claim 24, wherein the volume of the second cultivation bag is at least 150% of the volume of the first cultivation bag. 